Glutathione Oxidation by Hypochlorous Acid in Endothelial Cells Produces Glutathione Sulfonamide as a Major Product but Not Glutathione Disulfide (original) (raw)
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Oxidation of intracellular glutathione after exposure of human red blood cells to hypochlorous acid
Biochemical Journal, 1995
Exposure of human red blood cells to low doses of hypochlorous acid (HOCl) resulted in the loss of intracellular GSH. Oxidation occurred less than 2 min after the addition of HOCl, and required approx. 2.5 mol of HOCl per mol of GSH lost. Loss of GSH preceded oxidation of membrane thiols, the formation of chloramines and haemoglobin oxidation. The susceptibility of intracellular GSH to oxidation by HOCl was two-thirds that of GSH in cell lysates. These results indicate that HOCl can penetrate the red cell membrane, which provides little barrier protection for cytoplasmic components, and that GSH oxidation by HOCl may be a highly selective process. Virtually all of the GSH lost was converted into GSSG. If glucose was added to the medium, most of the GSH oxidized by low doses of HOCl was rapidly regenerated. At higher doses, recovery was less efficient. However, when HOCl was added as a slow infusion rather than in a single bolus, there was increased recovery at higher doses. This ind...
Hypochlorous acid inhibits glutathione S-conjugate export from human erythrocytes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2002
It was found that the hypochlorous acid (HOCl) inhibits the active efflux of glutathione S-conjugates, 2,4-dinitrophenyl-S-glutathione (DNP-SG, c 50% = 258 F 24 AM HOCl) and bimane-S-glutathione (B-SG, c 50% = 125 F 16 AM HOCl) from human erythrocytes, oxidises intracellular reduced glutathione (the ratio [HOCl]/[GSH] oxidized = 4) and inhibits basal as well as 2,4-dinitrophenol-(DNP) and 2,4dinitrophenyl-S-glutathione (DNP-SG)-stimulated Mg 2 + -ATPase activities of erythrocyte membranes. Multidrug resistance-associated protein (MRP1) mediates the active export of glutathione S-conjugates in mammalian cells, including human erythrocytes. A direct impairment of erythrocyte membrane MRP by hypochlorous acid was shown by electrophoresis and immunoblotting (c 50% = 478 F 36 AM HOCl). The stoichiometry of the MRP/HOCl reaction was 1:1. These results demonstrate that MRP can be one of the cellular targets for the inflammatory mediator hypochlorous acid. D
American Journal of Physiology-Heart and Circulatory Physiology, 1999
We investigated the effect of sublethal concentrations of hypochlorous acid (HOCl) on intracellular thiol groups. Exposure of human umbilical vein endothelial cells to HOCl caused a decrease in cell viability, with concentrations of ≤25 μM HOCl being sublethal. At these concentrations, we saw a loss of glutathione and total protein thiol groups. Of the thiol enzymes we investigated, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was particularly susceptible to inactivation, creatine kinase was moderately susceptible, and lactate dehydrogenase was unaffected by HOCl at the concentrations used. Similar results were obtained with HOCl generated over 30 min by myeloperoxidase. GAPDH activity could be regenerated on reincubation of cells in Hanks’ balanced salt solution or reduction with dithiothreitol. In contrast, glutathione loss was not reversible, and further decreased with time. Cellular ATP levels decreased with sublethal HOCl concentrations and this appeared to be unrelated to ...
The Biochemical journal, 1997
Reduced glutathione (GSH) is one of the most preferred biological substrates of myeloperoxidase-derived hypochlorous acid and is a likely target for neutrophil oxidants. We have used HPLC to show that the oxidation of GSH by hypochlorous acid gives two major, stable products in addition to glutathione disulphide (GSSG). The most prevalent product lacks free amine and thiol groups, and was shown by electrospray MS to have a molecular mass of 337 Da. This corresponds to GSH with a gain of two oxygen atoms and a loss of two hydrogen atoms, and is consistent with the product being an internal sulphonamide. The other novel product has a molecular mass of 644 Da, and has amine groups but no free thiols. These properties are consistent with it being glutathione thiolsulphonate. Whereas GSSG in the cell is recycled enzymically, formation of these higher oxidation products is likely to be irreversible. Hypochlorous acid, therefore, could compromise the cell by depleting GSH. The putative sul...
Lung, 1991
Exposure of cultured pulmonary artery endothelial cells to 95% 0 2 resulted in the following sequence of events: decrease in [3H]thymidine incorporation after 24 h; increase of intracellular glutathione (GSH) and loss of cellular protein after 48 h; increase of spontaneous and decrease of provoked prostacyclin formation as well as increased release of cellular LDH after 72 h. This oxygen toxicity model was used to study the following 2 questions. (1) What is the relative importance of the GSH redox Cycle compared to catalase as antioxidative defense against hyperoxia? Endothelial cells were grown in selenium-depleted medium to inhibit glutathione peroxidase activity. Endothelial GSH biosynthesis was inhibited by buthionine sulfoximine. Catalase activity was reduced by aminotriazole. Endothelial cells with an impaired GSH redox cycle were easily killed by hyperoxia within 24 h, while inhibition of catalase did not enhance the susceptibility of endothelial cells to hyperoxia. (2) Can endothelial GSH content be increased by exogenous sulfhydryl reagents and does this result in an increase of endothelial cells' resistance to hyperoxia? Exogenous GSH, N-acetylcysteine, cysteine, and L-2-oxothiazolidine-4-carboxylate (L-2-oxo) increased intracellular GSH. All sulfhydryl reagents (with the exception of L-2-oxo) protected endothelial cells from hyperoxia. Concentrations of exogenous GSH and N-acetylcysteine that did not increase intracellular GSH reduced hyperoxia-induced endothelial cell injury. Thus the capacity of the GSH redox cycle rather than intracellular GSH levels or catalase determines endothelial cells' resistance to hyperoxia.
Journal of Hepatology, 1991
The accumulation of oolymorphonuclear leucocytes (PMN) may pl?y an important role in liver injury by twins and ischemtaireperfusion. Upan activation there cells generate hypochlorcus acid <YOGI) and long-lived oxidants such as mor.ochloramine (NH2CI) and taurinxchioramine (TabNHCI) which auld r-wibute to organ injury when PMN accumulate in the liver. Therefore. the effectsnf HOCI, NH$l and Tau'JHCI on hepatic function were invcs:igrted in the perused rat liver. HOC1 at a concentxfion of 2.lyM resulted in a marked increase in the perfusion pressure atd the xlease of LDH associated wth a decrease in bile flaw. These effects were abolished by increasing&e *c*nowkdgnlents Supported by grar* No. 3.812-0.87 from rbe Swiss Nalional Foundation for Scientific Research and by Deut%he Fnrrchungsgemeinachaft. The technica, acsislance of Ms. E. Junker and Mr. c. Schraoz is gra*efuOy Xk".XVlZdg.Sd sinuroidal cndothcos, dls of lY0.s. tiwr ..h its modulation in
Molecules
Sulodexide (SDX), a purified glycosaminoglycan mixture used to treat vascular diseases, has been reported to exert endothelial protective effects against ischemic injury. However, the mechanisms underlying these effects remain to be fully elucidated. The emerging evidence indicated that a relatively high intracellular concentration of reduced glutathione (GSH) and a maintenance of the redox environment participate in the endothelial cell survival during ischemia. Therefore, the aim of the present study was to examine the hypothesis that SDX alleviates oxygen–glucose deprivation (OGD)-induced human umbilical endothelial cells’ (HUVECs) injury, which serves as the in vitro model of ischemia, by affecting the redox state of the GSH: glutathione disulfide (GSSG) pool. The cellular GSH, GSSG and total glutathione (tGSH) concentrations were measured by colorimetric method and the redox potential (ΔEh) of the GSSG/2GSH couple was calculated, using the Nernst equation. Furthermore, the leve...
Analysis of glutathione: implication in redox and detoxification
Clinica Chimica Acta, 2003
Background: Glutathione is a ubiquitous thiol-containing tripeptide, which plays a central role in cell biology. It is implicated in the cellular defence against xenobiotics and naturally occurring deleterious compounds, such as free radicals and hydroperoxides. Glutathione status is a highly sensitive indicator of cell functionality and viability. Its levels in human tissues normally range from 0.1 to 10 mM, being most concentrated in liver (up to 10 mM) and in the spleen, kidney, lens, erythrocytes and leukocytes. In humans, GSH depletion is linked to a number of disease states including cancer, neurodegenerative and cardiovascular diseases. The present review proposes an analysis of the current knowledge about the methodologies for measuring glutathione in human biological samples and their feasibility as routine methods in clinical chemistry. Furthermore, it elucidates the fundamental role of glutathione in pathophysiological conditions and its implication in redox and detoxification process. Tests available: Several methods have been optimised in order to identify and quantify glutathione forms in human biological samples. They include spectrophotometric, fluorometric and bioluminometric assays, often applied to HPLC analysis. Recently, a liquid chromatography-mass spectrometry technique for glutathione determination has been developed that, however, suffers from the lack of total automation and the high cost of the equipment. Conclusion: Glutathione is a critical factor in protecting organisms against toxicity and disease. This review may turn useful for analysing the glutathione homeostasis, whose impairment represents an indicator of tissue oxidative status in human subjects. D
Archives of Medical Science, 2019
Introduction: Sulodexide (SDX), a heparinoid used to treat vascular diseases, exerts anti-ischemic properties. However, the underlying molecular mechanisms remain unclear. Induction of glutathione (GSH)-dependent genes protects against ischemia. Here, we investigated the effect of SDX on GSH-associated gene expression in human umbilical endothelial cells (HUVECs) using an in vitro ischemia model. Material and methods: The transcriptional expression of GSH-related genes (GCLc, xCT, GS, GPx1 and GR) in HUVECs treated without/with SDX (0.5 LRU/ ml) under oxygen-glucose deprivation (OGD) condition for 1-6 h was analyzed by real-time polymerase chain reaction. Results: GCLc and xCT were strongly up-regulated by SDX in HUVECs in the first 2 h of OGD. GS and GPx1 mRNA expression levels were significantly increased during any time interval in ischemic HUVECs treated with SDX. Furthermore, incubation of HUVECs with SDX in OGD for 1-4 h resulted in enhanced expression of GR. Conclusions: Our studies provide the first evidence that SDX activates GSH-related genes in OGD-injured HUVECs.
Analytical and Bioanalytical Chemistry, 2007
A simple and highly effective reversed-phase (RP) high-performance liquid chromatography (HPLC) method is described for analysing glutathione (GSH) and glutathione disulfide (GSSG) in out-flowing supernatants and lysates of perfusion cell cultures of human kidney cells (HK-2 cells) continuously exposed to cadmium chloride (CdCl 2 ), which is a well-known nephrotoxin. The developed linear liquid chromatographic gradient employs monolithic poly(styrene-co-divinylbenzene) (PS/DVB) as a stationary phase and is adaptable for coupling to mass spectrometry via an electrospray ionisation interface (LC-ESI/MS), which is carried out in case of co-eluting peaks. This study presents a quantitative assay of glutathione over the time of experiment and cell lysates at the end of the experiment. The assay of out-flowing supernatants has the potential to be applied as an online assay in high time resolution. Glutathione (reduced and oxidised, GSH and GSSG) is chosen as an indicator for toxic effects in the cultured cells. In principle it is possible to show the concentration of glutathione as a function of time in an investigation of exposure of the HK-2 cell line to CdCl 2 . In addition to glutathione analysis, well-established assays of cell death such as enzyme release and cell viability are performed to obtain information about the number of living cells. Toxicity of 5 μM CdCl 2 is manifested in all of the assays applied. Fast (<7 min) and highly reproducible (max. aberration 4.7%) determination of glutathione could be achieved.